CN115698067A

CN115698067A - Cancer treatment using antibodies that bind LGR5 and EGFR

Info

Publication number: CN115698067A
Application number: CN202180037623.4A
Authority: CN
Inventors: 埃内斯托·伊萨克·沃瑟曼; 科内利斯·雅各布·约翰内斯·乔治·博尔; 绍博尔奇·法特劳伊
Original assignee: Merus BV
Current assignee: Merus BV
Priority date: 2020-04-24
Filing date: 2021-04-23
Publication date: 2023-02-03
Also published as: WO2021215926A1; BR112022021345A2; CN116731189A; EP4139357A1; TW202206460A; KR20230005281A; IL297443A; CA3176186A1; US20230192866A1; MX2022013182A; JP2023523006A; AU2021261681A1

Abstract

The present disclosure relates to means and methods for treating cancer. The present disclosure relates, inter alia, to a method of treating cancer in an individual using an antibody that binds LGR5 and EGFR. The invention further relates to combinations for use in such methods and combinations for the manufacture of a medicament for the treatment of gastrointestinal cancer. Such antibodies are particularly useful in the treatment of gastric, esophageal or gastro-esophageal junction cancer.

Description

Cancer treatment using antibodies that bind LGR5 and EGFR

Technical Field

Background

Traditionally, most cancer drug discovery has focused on drugs that block essential cell function and kill dividing cells via chemotherapy. Chemotherapy, however, rarely results in a complete cure. In most cases, tumors in patients stop growing or temporarily shrink (called remission) and as a result begin to proliferate again, sometimes more rapidly (called relapse) and become increasingly difficult to treat. Recently, the focus of cancer drug development has moved away from widespread cytotoxic chemotherapy to targeted cytostatic therapies with less toxicity. Targeted therapy with components that specifically inhibit the signaling pathway has been clinically validated in leukemia for the treatment of advanced cancers. However, in most cancers, the targeting approach still proves ineffective.

Despite many advances in disease treatment and increased awareness of the molecular events that lead to cancer, cancer remains a major cause of death worldwide. For example, gastric cancer is the 5 th most commonly diagnosed cancer worldwide, and is the 3 rd most fatal cancer. In 2018, 783,000 people were estimated to die of gastric cancer. Esophageal cancer is the 9 th most common cancer and the 6 th most common cause of cancer death. Epidermal Growth Factor Receptor (EGFR) has been reported to be overexpressed in more than 30% of cases of Gastric Adenocarcinoma (GAC) and Esophageal Adenocarcinoma (EAC). However, review of the analysis of six different studies concluded that the addition of anti-EGFR agents to chemotherapy did not improve the overall or progression-free survival of patients with advanced/metastatic EAC, GAC or gastro-esophageal junction adenocarcinoma (GEJAC) (Kim et al 2017 Oncotarget.2017, 11 months 17; 8 (58): 99033-99040). Thus, there is a need for the treatment of cancer, particularly gastric and esophageal cancer.

Disclosure of Invention

The present disclosure provides the following preferred embodiments. However, the present invention is not limited to these embodiments.

In some embodiments, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in treating cancer in a subject, wherein the use comprises providing the subject with a 1500mg fixed dose (flat dose) of the antibody or functional portion, derivative and/or analog thereof. The present disclosure further provides a method of treating cancer in a subject, comprising providing a fixed dose of 1500mg of the antibody or functional part, derivative and/or analogue thereof to a subject in need thereof.

In some embodiments, the present disclosure provides an antibody, or functional portion, derivative and/or analog thereof, comprising a variable domain that binds an extracellular portion of EGFR and a variable domain that binds an extracellular portion of LGR5 for use in treating gastric, esophageal, or gastro-esophageal junction cancer in a subject. The present disclosure further provides a method of treating gastric, esophageal, or gastro-esophageal junction cancer in a subject comprising providing an antibody or functional portion, derivative and/or analog thereof to a subject in need thereof. Preferably, the use comprises providing a fixed dose of 1500mg of the antibody or functional part, derivative and/or analogue thereof to the subject.

In some embodiments, the administration of the therapeutic compound can be performed weekly, biweekly, or monthly. In some embodiments, the therapeutic compound is administered once every 2 weeks.

In some embodiments, the present disclosure provides an antibody, or a functional portion, derivative and/or analog thereof, comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in treating gastric, esophageal, or gastro-esophageal junction cancer in a Her2 negative subject. The present disclosure further provides a method of treating gastric, esophageal, or gastro-esophageal junction cancer in a Her 2-negative subject, comprising providing an antibody or functional part, derivative and/or analog thereof to a subject in need thereof. Preferably, the use comprises providing a fixed dose of 1500mg of the antibody or functional part, derivative and/or analogue thereof to the subject. In some embodiments, administration of a therapeutic compound to a Her2 negative subject can be performed weekly, biweekly, or monthly. Preferably, the therapeutic compound is administered once every 2 weeks.

Preferably, the antibody or functional part, derivative and/or analogue thereof is provided intravenously.

Preferably, the cancer has a mutation in one or more genes selected from: TP53, MLH1, PIK3CA, CDKN2A, UGT1A8, BRAF, PTEN and KRAS, preferably wherein the cancer has a mutation in one or more genes selected from: TP53, MLH1, CDKN2A, UGT1A8, BRAF and PTEN. Preferably, the cancer has one or more mutations selected from: TP 53R 196T; TP 53R 342T; TP 53R 248Q; MLH 1V 384D; PIK3CA H1047R; CDKN2A W110T; UGT1A1G71R; UGT1 A8G 71R; and KRAS G12C.

Preferably, the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R196T. Preferably, the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably wherein the mutation is V384D.

Preferably, the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R248Q; the cancer has a mutation in the gene encoding PIK3CA, preferably wherein the mutation is H1047R; the cancer has a mutation in a gene encoding CDKN2A, preferably wherein the mutation is W110T; the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R.

Preferably, the cancer is esophageal cancer, preferably Esophageal Squamous Cell Carcinoma (ESCC).

Preferably, the cancer has a mutation in the gene encoding BRAF. Preferably, however, the cancer does not have the mutation V600E in BRAF, and wherein the cancer has a mutation in the gene encoding PTEN. Preferably, however, the cancer also does not have the mutation R130Ter in PTEN.

Preferably, the cancer has a mutation in the gene encoding KRAS, preferably wherein the mutation is G12C; the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R.

Preferably, the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R. Preferably, the cancer further has a mutation in PIK3CA, preferably wherein the mutation is E545K.

Preferably, the cancer is gastric cancer.

Preferably, the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in figure 3; or an amino acid sequence having at most 15, preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and preferably no more than 5, 4, 3, 2 or 1 amino acid modifications, including insertions, deletions, substitutions or combinations thereof, relative to the VH in a VH chain MF3755 as depicted in figure 3; and wherein the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in figure 3; or an amino acid sequence having up to 15, preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and preferably no more than 5, 4, 3, 2 or 1 amino acid modifications, including insertions, deletions, substitutions or combinations thereof, relative to the VH in a VH chain MF5816 as depicted in figure 3.

Preferably, the variable domain that binds LGR5 binds to an epitope located within amino acid residues 21-118 of the human LGR5 sequence depicted in figure 1. Preferably, the amino acid residues at positions 43, 44, 46, 67, 90 and 91 of human LGR5 are involved in the binding of the LGR5 binding variable domain to LGR5. Preferably, the LGR5 binding variable domain binds weakly to LGR5 proteins comprising one or more of the mutations in amino acid residues selected from 43A, 44A, 46A, 67A, 90A and 91A.

Preferably, the variable domain that binds EGFR binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence depicted in figure 2. Preferably, the amino acid residues at positions I462, G465, K489, I491, N493 and C499 of human EGFR are involved in the binding of the EGFR binding variable domain to EGFR. Preferably, the EGFR binding variable domain binds weakly to EGFR proteins comprising one or more of the amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A and C499A.

Preferably, the antibody is enhanced by ADCC. Preferably, the antibody is non-fucose modified (afucosylated).

Drawings

FIG. 1: a human LGR5 sequence; sequence ID NO:1.

FIG. 2: a human EGFR sequence; sequence ID NO:2.

fig. 3 a): the amino acid sequence of the heavy chain variable region (sequence ID Nos: 3-15), which together with a common light chain variable region (e.g., human kappa light chain IgV kappa 139 x 01/IGJ kappa 1 x 01) forms a variable domain that binds LGR5 and EGFR. The CDRs and framework regions are indicated in fig. 3 b). The respective DNA sequences are indicated in fig. 3 c).

A of fig. 4): an amino acid sequence of a common light chain amino acid sequence. b) The method comprises the following steps DNA sequence and translation of the common light chain variable region (IGKV 1-39/jk 1). c) The method comprises the following steps Light chain constant region DNA sequence and translation. d) The method comprises the following steps V region IGKV1-39A; e) The method comprises the following steps CDR1, CDR2, and CDR3 of the common light chain according to IMGT numbering.

FIG. 5: igG heavy chain for the production of bispecific molecules. a) CH1 region DNA sequence and translation. b) Hinge region DNA sequence and translation. c) CH2 region DNA sequence and translation. d) A CH3 domain containing mutant L351K and T366K (KK) DNA sequences and translation. e) The CH3 domain and translation of DNA sequences containing mutations L351D and L368E (DE). Residue positions are according to EU numbering.

FIG. 6: data show mean tumor size in a) gastric PDX model and b) esophageal PDX model with SEM error bars. Statistical significance at a given time point was calculated using a Two-factor variational analysis (Two-way ANOVA) assay. ADC = adenocarcinoma. SCC = squamous cell carcinoma. The grey area indicates the treatment period.

Detailed Description

In order that the present specification may be more readily understood, certain terms are first defined. Other definitions are set forth throughout the embodiments. Unless defined separately herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and employ conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology.

As used herein, the singular forms "a", "an" and "the" include plural references. The use of the terms "comprising", "having", "including" and other forms, such as "comprising", "having" and "including", is not limiting.

As used herein, the term "antibody" means a protein molecule belonging to the class of immunoglobulins that contains one or more domains that bind to an epitope on an antigen, where such domains are or are derived from or have sequence homology to the variable region of an antibody. Antibodies are typically composed of basic building blocks-each basic building block having two heavy chains and two light chains. The antibody according to the present invention is not limited to any particular form or method of producing the same.

A "bispecific antibody" is an antibody as described herein, wherein one domain of the antibody binds to a first antigen and a second domain of the antibody binds to a second antigen, wherein the first antigen and the second antigen are not identical, or wherein one domain binds to a first epitope on an antigen and the second domain binds to a second epitope on an antigen. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region/light chain variable region (VH/VL) combination binds a first antigen or a first epitope on an antigen, and a second VH/VL combination binds a second antigen or a second epitope on an antigen. The term further includes antibodies in which a VH is capable of specifically recognizing a first antigen and a VL paired with a VH in an immunoglobulin variable region is capable of specifically recognizing a second antigen. The resulting VH/VL pair will bind antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO 2010/108127 and Schaefer et al (Cancer Cell 20,472-486, 10 months 2011). Bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of production thereof.

As used herein, "common light chain" refers to the two light chains (or VL portions thereof) in a bispecific antibody. The two light chains (or VL portions thereof) may be identical or have some amino acid sequence differences, while the binding specificity of the full-length antibody is unaffected. The terms "common light chain," "common VL," "single light chain," and "single VL" are used interchangeably herein, with or without the addition of the term "rearrangement". "common" also refers to functional equivalents of light chains that are not identical in amino acid sequence. There are many variants of this light chain in which there are mutations (deletions, substitutions, insertions and/or additions) that do not affect the formation of functional binding regions. The light chain of the invention may also be a light chain as specified herein, having from 0 to 10, preferably from 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof. For example, light chains that are made or found to be non-identical, but still functionally equivalent, such as by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute, or contribute only in part to binding specificity when paired with a heavy chain, and the like, fall within the definition of a common light chain as used herein.

As used herein, "comprise" and its inflectional forms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "to consist of" \8230; \8230, the composition may be replaced by "consisting essentially of" \8230; \8230, the composition "means that the compound or adjuvant compound as defined herein may comprise additional component(s) in addition to the specifically identified component(s), which additional component(s) do not alter the unique characteristics of the present invention.

The term "full-length IgG" or "full-length antibody" according to the invention is defined to encompass essentially whole IgG, however it does not necessarily have all the functions of whole IgG. For avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains designated CH1, CH2, CH3, VH and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion and, after binding, can interact with molecules and cells of the immune system via the constant domains (mainly via the Fc portion). Full length antibodies according to the invention encompass IgG molecules in which mutations may be present that provide the desired characteristics. Full-length iggs should not have deletions of substantial portions of either region. However, igG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule are encompassed within the term "full-length IgG". For example, such IgG molecules may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR regions, wherein the deleted amino acids are not essential for the antigen binding specificity of the IgG.

"derivatives of antibodies" are proteins that deviate from the amino acid sequence of a natural antibody by up to 20 amino acids, except for the CDR regions. Derivatives of an antibody as disclosed herein are antibodies that deviate from the amino acid sequence by up to 20 amino acids.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to residues in the selected sequence after the sequences are aligned for optimal comparison purposes. Comparison of percent sequence identity of nucleic acid sequences Vector NTI was used

The AlignX application of the software 11.5.2, was determined using default settings determined using the modified ClustalW algorithm (Thompson, J.D., higgins, D.G., and Gibson T.J., (1994) Nuc.acid Res.22 (22): 4673-4680), swgapdpamt score matrix, gap open penalty 15, and gap extension penalty 6.66. Amino acid sequence utilization of Vector NTI

The AlignX application of the software 11.5.2, aligned using default settings, which were adjusted using the modified ClustalW algorithm (Thompson, J.D., higgins, D.G., and Gibson T.J., (1994) Nuc.acid Res.22 (22): 4673-4680), the blosum62mt2 score matrix, the gap opening penalty of 10, and the gap extension penalty of 0.1.

Since antibodies typically recognize epitopes of an antigen, and such epitopes may also be present in other compounds, an antibody according to the invention that "specifically recognizes" an antigen, such as EGFR or LGR5, may also recognize other compounds if such other compounds contain epitopes of the same species. Thus, the term "specifically recognizes" with respect to an antigen and antibody interaction does not exclude the binding of an antibody to other compounds containing the same kind of epitope.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids that are adjacent due to tertiary folding of the protein (so-called linear epitopes and conformational epitopes). Epitopes formed by consecutive linear amino acids are generally retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding, conformation are generally lost after treatment with denaturing solvents. An epitope can typically include 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial configuration.

As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse pig, and the like (e.g., a patient suffering from cancer, such as a human patient).

As used herein, the term "treating" refers to any type of intervention or method performed on a subject, or the administration of an active agent or combination of active agents to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of the symptoms, complications, conditions, or biochemical markers associated with the disease.

As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that produces a beneficial effect, such as an improvement in at least one symptom of a disease or disorder (e.g., cancer). The beneficial effect may be in the form of an improvement relative to baseline, including an improvement relative to measurements or observations made prior to initiation of therapy according to the method. For example, the beneficial effect may be in the form of slowing, stabilizing, stopping, or reversing the progression of cancer in the subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or cancer markers. Effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or arrest tumor growth, and/or prevent or delay tumor recurrence or recurrence.

The term "effective amount" or "therapeutically effective amount" refers to the amount of a drug or combination of drugs that provides the desired biological, therapeutic and/or prophylactic result. The result can be a reduction, amelioration, palliation, alleviation, delay and/or remission of one or more of the signs, symptoms or causes of a disease, or any other desired alteration of a biological system. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. The effective amount of the drug or composition may be: (ii) (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) Inhibit, block, slow down and stop cancer cell infiltration to peripheral organs to a certain extent; (iv) inhibition of tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is an EGFR/LGR5 antibody that achieves a reduction in cancer (e.g., a reduction in the number of cancer cells); an amount that slows progression of the cancer or prevents regeneration or recurrence of the cancer.

The present disclosure provides antibodies, or functional portions, derivatives and/or analogs thereof, comprising a variable domain that binds an extracellular portion of EGFR and a variable domain that binds an extracellular portion of LGR5 for use in treating cancer. The words cancer and tumor are used herein and, unless specifically stated otherwise, refer generally to cancer.

Epidermal Growth Factor (EGF) receptors (EGFR, erbB1 or HER 1) are members of the four-Receptor Tyrosine Kinase (RTK) family designated HER-or cErbB-1, -2, -3 and-4. EGFR is known as various synonyms, the most common of which is EGFR. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and two in homo-and heterodimerization. EGFR integrates extracellular signals from various ligands to generate diverse intracellular responses. The major signal transduction pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by recruitment of Grb2 to tyrosine phosphorylated EGFR. This results in Ras activation via Grb2 binding to the heptad (Son of Sevenless, SOS) Ras-guanine nucleotide exchange factor. In addition, the PI 3-kinase-Akt signal transduction pathway is also activated by EGFR, but this activation is much stronger in the presence of ErbB-3 (HER 3) co-expression. EGFR is implicated in several human epithelial malignancies, in particular breast cancer, bladder cancer, non-small cell lung cancer, colorectal cancer, ovarian cancer, head and neck cancer and brain cancer. Activating mutations in genes and overexpression of receptors and their ligands have been found, which lead to autocrine activation loops. Therefore, this RTK has been widely used as a target for cancer therapy. Both small molecule inhibitors targeting RTKs and monoclonal antibodies (mabs) directed against extracellular ligand-binding domains have been developed and have thus far shown several clinical successes, even though most select groups of patients have. The database registration number of the human EGFR protein and the coding gene thereof is GenBank NM-005228.3. This accession number is given primarily to provide another means of identifying the EGFR protein of interest, and the actual sequence of the EGFR protein to which the antibody binds may vary, for example due to coding gene mutations, such as those found in some cancers or the like.

Unless otherwise indicated, where reference is made herein to EGFR, reference refers to human EGFR. The variable domain antigen binding site that binds EGFR and its various variants, such as those expressed on some EGFR-positive tumors.

The term "LGR" refers to a family of proteins called G protein-coupled receptors containing leucine-rich repeats. Several members of this family are known to participate in the WNT signaling pathway, notably LGR4; LGR5 and LGR6.

LGR5 is a G protein-coupled receptor 5 containing leucine-rich repeats. An alternative name for a gene or protein is G protein-coupled receptor 5 containing leucine-rich repeats; g protein-coupled receptor 5 containing leucine-rich repeats; g protein-coupled receptor HG38; g protein-coupled receptor 49; a G protein-coupled receptor 67; GPR67; GPR49; orphan G protein-coupled receptor HG38; g protein-coupled receptor 49; GPR49; HG38 and FEX. The protein or antibody of the present invention that binds LGR5 binds to human LGR5. Due to sequence and tertiary structure similarities between humans and other mammalian xenologues, LGR5 binding proteins or antibodies of the invention may also, but need not, bind to such xenologues. The database accession numbers for the human LGR5 protein and its coding gene are (NC — 000012.12, nt_029419.13, np_018923.2, np _001264155.1, np _001264156.1. The accession numbers are given mainly in order to provide another method of identifying LGR5 as a target, and the actual sequence of the LGR5 protein bound may vary, for example, due to coding gene mutations, such as those occurring in some cancers or the like. The LGR5 antigen-binding site binds LGR5 and its various variants, such as those expressed by some LGR 5-positive tumor cells.

In some embodiments, the cancer is a gastrointestinal cancer, for example, a colorectal cancer. Preferably, the cancer is gastric cancer, esophageal cancer, or gastro-esophageal junction cancer. Gastric cancer (gastric cancer/stomach cancer) is a cancer that develops from the gastric mucosa and in particular from the mucus-producing glandular cells found therein. Such cancers are also referred to as adenocarcinomas, or gastric adenocarcinomas in this case, as they develop from the gastric mucosa. In a preferred embodiment, the cancer is therefore gastric adenocarcinoma or a cancer that develops from the gastric mucosa, the terms being used interchangeably herein. Esophageal cancer is a cancer that develops from the esophagus. The two major secondary types are Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC). Gastro-esophageal junction cancer (also known as gastro-esophageal junction adenocarcinoma) originates from the gastro-esophageal junction.

In some embodiments, the cancer expresses LGR5 and/or expresses EGFR. As used herein, a cancer expresses LGR5 if the cancer comprises LGR 5-expressing cells. LGR 5-expressing cells contain detectable levels of LGR 5-encoding RNA. As used herein, a cancer expresses EGFR if the cancer comprises cells that express EGFR. Cells expressing EGFR contain detectable levels of RNA encoding LGR5. Expression can also be detected by incubating the cells with an antibody that binds to LGR5 or EGFR. However, some cells do not express proteins in high enough amounts for such antibody tests. In such cases, mRNA or other forms of nucleic acid sequence detection are preferred.

In some embodiments, the present disclosure provides an antibody or functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in treating gastric, esophageal or gastro-esophageal junction cancer in a subject whose Her2 status is selected from Her2 positive, her2 high, her 23 +, her 2+, her 1+, her 20 or Her2 negative subject. Preferably, the subject is Her2 negative. The present disclosure further provides a method of treating gastric, esophageal, or gastro-esophageal junction cancer in a Her 2-negative subject, comprising providing an antibody or functional part, derivative and/or analog thereof to a subject in need thereof. Preferably, the use comprises providing a fixed dose of 1500mg of the antibody or functional part, derivative and/or analogue thereof to the subject. In some embodiments, administration of a therapeutic compound to a Her2 negative subject can be performed weekly, biweekly, or monthly. Preferably, the therapeutic compound is administered once every 2 weeks.

Methods for determining the expression of human epidermal growth factor receptor 2 (HER 2) in a subject are well known in the art. For example, expression levels of Her2 can be established using Immunohistochemistry (IHC) or (fluorescence) In Situ Hybridization (ISH), which allows for identification of Her2 status, including identification of Her2 negative subjects. IHC or ISH are both well-defined and standard procedures routinely used to establish Her2 status in human subjects. Reference is made herein to the ASCO/CAP guidelines, for example, according to Bartley et al (HER 2 Testing and Clinical Decision Making in Gastroesophageal Adenociarco. Arch Pathol Lab. Med.2016; 140: 1345-1363). For example, the use of an anti-HER-2/neu antibody (clone 4B 5) allows for semi-quantitative detection of HER-2 antigen in sections of FFPE gastric/gastroesophageal adenocarcinoma, gastric cancer, esophageal cancer, or gastro-esophageal junction cancer using IHC. Staining and scoring were performed according to consensus guidelines for this cancer type. Such IHC tests typically provide a score of 0 to 3+, which measures the amount of HER2 receptor protein on the cell surface in cancer tissue samples. Based on IHC scores, patients can be classified as Her2 negative, e.g., when a score of 0 or 1+ is measured. If ISH testing is used to establish Her2 expression, e.g. using Her2 probe (17q11.2-q 12) and centromere 17 probe (Cen 17), then a diagnosis is "positive" or "negative", sometimes also reported as Her2 being "zero". The treatment methods of the present disclosure are subjects preferably established as Her2 negative by virtue of IHC and/or ISH.

Herein, a Her 2-negative subject means a subject having a Her 2-negative cancer, cancer cell or tumor. Her2 status may be determined as described above according to IHC and/or ISH.

Preferably, in some embodiments, the treatment with the antibody or functional part, derivative and/or analogue thereof is preceded by a step of diagnosing Her2 status in the subject. Preferably, in some embodiments, a subject with Her2 negative status is selected for treatment. Preferably, in some embodiments, the treating the subject is preceded by the step of diagnosing the subject as having Her2 negative gastric cancer, esophageal cancer, or gastro-esophageal junction cancer. Such cancers treated by the methods of the present disclosure include gastric adenocarcinoma and esophageal cancer with squamous cell carcinoma histology.

The Her2 negative diagnosis preferably involves an ISH or IHC test on Her2 status.

Preferably, in some embodiments, treating Her2 negative subjects is preceded by the step of screening for subjects having Her2 negative gastric, esophageal, or gastro-esophageal junction cancer. Such cancers are especially adenocarcinomas. The screening preferably involves performing an ISH or IHC test on Her2 status.

Cancer, such as gastric cancer, esophageal cancer, or cancer of the gastro-esophageal junction may be associated with the presence of mutations. Such mutations include mutations in known oncogenes, such as PIK3CA, KRAS and BRAF. Oncogenic mutations are generally described as activating mutations or mutations that produce new functions. Another type of cancer mutation involves tumor suppressor genes, such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are generally inactive.

TP53 encodes a transcription factor that regulates a variety of activities including stress response and cell proliferation. Mutations in TP53 are associated with various cancers and are estimated to occur in more than 50% of human cancers, including gastric and esophageal cancers. Specifically, the TP 53R 248Q mutation was shown to be associated with cancers including gastric and esophageal cancers (Pitolli et al int.j.mol.sci.20120. Nonsense mutations at positions R196 and R342 have been identified in a variety of tumors, such as from the breast and esophagus, respectively; and tumors of the ovary, prostate, breast, pancreas, stomach, large intestine/rectum, lung, esophagus, bone (Priestly et al Nature 2019 575. In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having TP53 mutations, particularly mutations that result in reduced TP53 expression or activity.

MutL homolog 1 (MLH 1) encodes a protein involved in DNA mismatch repair and is a known tumor suppressor gene. Mutations in MLH1 are associated with various cancers including gastrointestinal cancers. Low levels of MLH1 are also associated with esophageal cancer patients with a family history of esophageal cancer (Chang et al Oncol Lett.20159: 430-436), and MLH1 mutations in 1.39% of patients with malignant esophageal tumors (The AACR Project GENIE Consortium. AACR Project GENIE: a powering prediction medicine through an international consortium. Cancer discovery.2017;7 (8): 818-831. Data set 6 th edition). In particular, the MLH 1V 384D mutation was shown to be associated with cancer, such as colorectal cancer (Ohsawa et al Molecular Medicine Reports 2009 2: 887-891). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having MLH1 mutations, particularly mutations that result in reduced MLH1 expression or activity.

Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK 3 CA) encodes the 110kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3K). Mutations in PIK3CA are associated with various cancers, including gastrointestinal cancers. According to American Cancer Research institute (American Association for Cancer Research), 12.75% of patients with malignant solid tumors have reported a PIK3CA mutation. Specifically, the PIK3CA H1047R mutation was present in 2.91% of all patients with malignant solid tumors, and PIK3CA E545K was present in 2.55% of all patients with malignant solid tumors (see The AACR Project GENIE Consortium. AACR Project GENIE: power compression media through an international consortium. Cancer discovery.2017 (8): 818-831. Dataset 6 th edition). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having PIK3CA mutations, particularly oncogenic mutations in PIK2 CA.

The cyclin-dependent kinase inhibitor 2A (CDKN 2A) encodes a protein that inhibits CDK4 and ARF. According to the american society for cancer research reports CDKN2A mutations in 22.21% of patients with esophageal cancer, 28.7% of patients with esophageal squamous cell carcinoma, and 6.08% of patients with gastric adenocarcinoma. In particular, the CDKN2AW110Ter mutation is present in approximately 0.11% of cancer patients. (The AACR Project GENIE Consortium. AACR Project GENIE: power reduction media through an international contract. Cancer discovery.2017;7 (8): 818-831. Dataset 6 th edition). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having CDKN2A mutations, particularly mutations that result in reduced CDKN2A expression or activity.

The phosphatase and tensin homolog (PTEN) encodes

phosphatidylinositol

3,4, 5-triphosphate 3-phosphatase. PTEN mutations have been reported by the american society for cancer research in 6.28% of cancer patients, 3.41% of patients with gastric adenocarcinoma, 2.37% of patients with esophageal carcinoma, and 2.22% of patients with esophageal adenocarcinoma. Specifically, the PTEN R130Ter mutation (where Ter refers to a termination/stop codon) is present in 0.21% of all colorectal cancer patients (The AACR Project GENIE Consortium. AACR Project GENIE: power prediction medium through an international consortium. Cancer discovery.2017;7 (8): 818-831. Data set 6 th edition). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having PTEN mutations, particularly mutations that result in reduced expression or activity of PTEN.

BRAF encodes the serine/threonine protein kinase B-Raf, which is involved in growth signaling. According to the american cancer research association, BRAF mutations are reported in 1.91% of patients with gastric carcinoma and 1.93% of patients with gastric adenocarcinoma. Specifically, the BRAF V600E mutation is present in 2.72% of cancer patients (see The AACR Project GENIE Consortium. AACR Project GENIE: power prediction media through an international consensus cancer. Cancer discovery.2017;7 (8): 818-831. Data set 6 th edition). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancer with BRAF mutations, particularly oncogenic mutations in BRAF. However, in some embodiments, the therapeutic compounds disclosed herein are suitable for treating gastric cancer without the BRAF mutation V600E.

The Kirsten RAt Sarcoma (KRAS) encodes proteins that are part of the RAS/MAPK pathway. KRAS mutations were reported by The American society for research on cancer, in 14.7% of patients with malignant solid tumors, with KRAS G12C present in 2.28% of all patients with malignant solid tumors (see The AACR Project GENIE Consortium. AACR Project GENIE: power precision media needle international consortium. Cancer discovery.2017 (8): 818-831. Data set 6 th edition). In some embodiments, the therapeutic compounds disclosed herein are suitable for treating cancer having KRAS mutations, particularly oncogenic mutations in KRAS.

Uridine diphosphate glucuronosyltransferase 1A1 (UGT 1 A1) and uridine diphosphate glucuronosyltransferase 1A8 (UGT 1 A8) encode enzymes of the glucuronidation pathway. Several polytypes of reduced enzymatic activity are known to affect the metabolism and efficacy of irinotecan (irinotecan). For example, UGT1A1 × 6 duality genes (G71R polytype phenomenon) have a duality gene frequency of about 0.13% in chinese, korean and japanese populations, and UGT1A1 × 28 duality genes (dinucleotide repeat polytype phenomenon in TATA sequence of promoter region) are risk factors for irinotecan-induced neutropenia. In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having UGT1A1 and/or UGT1A8 mutations, particularly mutations that result in a reduction in UGT1A1 and/or UGT1A8 expression or activity.

Telangiectasia dysregulated muteins (ATM) are members of the serine-threonine kinase family and coordinate cellular responses to DNA damage via activation of unique DNA repair and signaling pathways. ATM germline mutations are associated with telangiectasia disorders, and ATM somatic mutations are commonly observed in endometrial, colorectal, pancreatic, breast, and urothelial cancers.

In preferred embodiments, the disclosure provides methods for treating cancer having a mutation in a gene encoding TP53, MLH1, PIK3CA, CDKN2A, UGT1A8, BRAF, PTEN, and KRAS. Preferably, the cancer has one or more mutations selected from the group consisting of: TP 53R 196T; TP 53R 342T; TP 53R 248Q; MLH 1V 384D; PIK3CA H1047R; PIK3CAE545K; CDKN2AW110T; UGT1A1G71R; UGT1 A8G 71R; and KRAS G12C. In some embodiments, the cancer is KRAS wild-type. Alternatively, the present disclosure provides methods for treating cancer having a mutation in the gene encoding ATM, particularly mutation W57T. In particular, the present disclosure provides methods for treating esophageal cancer, particularly ESCC, having a mutation in the gene encoding ATM, particularly mutation W57T.

In some embodiments, the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably wherein the mutation is V384D.

In some embodiments, the cancer has a mutation in a gene encoding TP53, preferably wherein the mutation is R248Q; the cancer has a mutation in the gene encoding PIK3CA, preferably wherein the mutation is H1047R; the cancer has a mutation in a gene encoding CDKN2A, preferably wherein the mutation is W110T; the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R. Preferably, the cancer is esophageal cancer, preferably Esophageal Squamous Cell Carcinoma (ESCC).

In some embodiments, the cancer has a mutation in the gene encoding BRAF. However, the cancer preferably does not have the mutation V600E in the gene encoding BRAF, and preferably does not have the mutation R130Ter in the gene encoding PTEN. In some embodiments, the cancer has a mutation in the gene encoding KRAS, preferably wherein the mutation is G12C; the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R. In some embodiments, the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R. In some embodiments, the cancer has a mutation in PIK3CA, preferably wherein the mutation is E545K. Preferably, the cancer is gastric cancer.

An antibody or functional part, derivative and/or analogue thereof as described herein comprises a variable domain that binds to an extracellular portion of an Epidermal Growth Factor (EGF) receptor and a variable domain that binds to LGR5. The EGFR is preferably human EGFR. LGR5 is preferably human LGR5. An antibody or functional part, derivative and/or analogue thereof as described herein comprises a variable domain that binds to an extracellular portion of a human Epidermal Growth Factor (EGF) receptor and a variable domain that binds to human LGR5.

Preferably, the antibody or functional part, derivative and/or analogue thereof as described herein comprises a variable domain that binds to an extracellular portion of an Epidermal Growth Factor (EGF) receptor and interferes with EGF binding to the receptor and a variable domain that binds LGR5, wherein the interaction of the antibody with LGR5 on LGR 5-expressing cells does not block the binding of R-spondin (RSPO) to LGR5. Methods for determining whether an antibody blocks or does not block binding of R-vertebrate protein to LGR5 are described in WO2017069528, which is incorporated herein by reference.

When the accession numbers or alternative names for proteins/genes are given herein, which are given primarily to provide another means of identifying the mentioned proteins as targets, the actual sequence of the target protein to which the antibodies of the invention bind may vary, for example due to mutations and/or alternative splicing in the encoding gene, such as those that occur in some cancers or similar diseases thereof. The protein of interest is bound by the antibody, so long as the epitope is present in the protein and the epitope is accessible to the antibody.

The antibody or functional part, derivative and/or analogue thereof as described herein preferably interferes with the binding of the ligand of EGFR to EGFR. As used herein, the term "interfere with binding" means that the binding of the antibody or functional part, derivative and/or analogue thereof to EGFR competes with the ligand for binding to the EGF receptor. The antibody or functional part, derivative and/or analogue thereof may attenuate ligand binding, displace the ligand when it has bound to the EGF receptor, or it may at least partially prevent the ligand from being able to bind to the EGF receptor, e.g. via steric hindrance.

The EGFR antibodies as disclosed herein preferably inhibit EGFR ligand-induced signaling, measured as ligand-induced growth of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or ligand-induced cell death of a431 cells (ATCC CRL-1555), respectively. EGFR can bind to a variety of ligands and stimulate the growth of the referred BxPC3 cells or BxPC3-luc2 cells. The growth of BxPC3 or BxPC3-luc2 cells is stimulated in the presence of EGFR ligand. EGFR ligand-induced BxPC3 cell growth can be measured by comparing cell growth in the absence and presence of ligand. Preferably the EGFR ligand used to measure EGFR ligand induced growth of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced growth is preferably measured using a saturating amount of ligand. In a preferred embodiment, EGF is used in an amount of 100ng/ml of medium. The EGF is preferably EGF R & D systems, cat Nos. 396-HB and 236-EG (see also WO2017/069628; incorporated herein by reference).

The EGFR antibody as disclosed herein preferably inhibits EGFR ligand-induced growth of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR can bind to a variety of ligands and stimulate the growth of the referred BxPC3 cells or BxPC3-luc2 cells. Growth of BxPC3 or BxPC3-luc2 cells was stimulated in the presence of ligand. EGFR ligand-induced BxPC3 cell growth can be measured by comparing cell growth in the absence and presence of ligand. Preferably the EGFR ligand used to measure EGFR ligand induced growth of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced growth is preferably measured using a saturating amount of ligand. In a preferred embodiment, EGF is used in an amount of 100ng/ml of medium. The EGF is preferably R & D systems, cat Nos. 396-HB and 236-EG (see also WO2017/069628; incorporated herein by reference).

For the avoidance of doubt, reference to cell growth as used herein refers to a change in the number of cells. Growth inhibition refers to a reduction in the number of cells that would otherwise be obtained. An increase in growth refers to an increase in the number of cells that would otherwise be obtained. Cell growth generally refers to cell proliferation.

Whether an antibody as described herein inhibits signaling or inhibits growth in a multispecific form is preferably determined by means of a method as described above using a monospecific monovalent or monospecific bivalent form of the antibody. Such antibodies preferably have a binding site that signals the receptor to be identified. Monospecific monovalent antibodies may have variable domains with unrelated binding specificities, e.g., tetanus toxoid specificity. Preferably the antibody is a bivalent monospecific antibody wherein the antigen binding variable domain consists of a variable domain that binds to a member of the EGF receptor family.

Merus is in it

Antibodies were planned to develop multispecific antibodies targeting EGFR and LGR5 (G protein-coupled receptors containing leucine-rich repeats). The efficacy of such multispecific antibodies has been assessed in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, e.g., WO2017/069628; incorporated herein by reference). Multispecific antibodies targeting EGFR and LGR5 were shown to inhibit tumor growth. The efficacy of such inhibitory antibodies was shown to correlate with LGR5 RNA expression levels of cells from cancer. Multispecific antibodies targeting EGFR and LGR5 as described in WO2017/069628 are particularly preferred.

An antibody or functional part, derivative and/or analogue thereof as described herein comprises a variable domain that binds an extracellular portion of LGR5. The variable domain that binds the extracellular portion of LGR5 preferably binds an epitope located within amino acid residues 21-118 of the sequence of figure 1, amino acid residue D43; g44, M46, F67, R90 and F91 are involved in binding of the antibody to the epitope.

LGR5 variable domains are preferably variable domains in which amino acid residues in LGR5 are substituted for D43A; one or more of G44A, M46A, F67A, R90A, and F91A attenuates binding of the variable domain to LGR5.

The epitope on the extracellular portion of LGR5 is preferably located within amino acid residues 21-118 of the sequence of figure 1. Preferably it is an epitope wherein the binding of the LGR5 variable domain to LGR5 is attenuated by one or more of the following amino acid residue substitutions in LGR 5: D43A; G44A, M46A, F67A, R90A, and F91A.

The present disclosure further provides antibodies having a variable domain that binds an extracellular portion of EGFR and a variable domain that binds an extracellular portion of LGR5, wherein the LGR5 variable domain binds an epitope on LGR5 that is within amino acid residues 21-118 of the sequence of figure 1.

The epitope on LGR5 is preferably a conformational epitope. The epitope is preferably located within amino acid residues 40-95 of the sequence of figure 1. The binding of the antibody to LGR5 is preferably attenuated by one or more of the following amino acid residue substitutions: D43A; G44A, M46A, F67A, R90A, and F91A.

Without being bound by theory, it is believed that M46, F67, R90 and F91 of LGR5 as depicted in figure 1 are contact residues of the variable domain as indicated above, i.e. the antigen binding site at which the variable domain binds an epitope of LGR5. Amino acid residue substitutions D43A and G44A to attenuate antibody binding may be due to these residues also being contact residues, however, it is also possible that these amino acid residue substitutions induce a (slight) modification of the conformation of the portion of LGR5 having one or more of the other contact residues (i.e. at positions 46, 67, 90 or 91), and that the conformational change results in attenuated antibody binding. Epitopes are characterized by the amino acid substitutions mentioned. Whether an antibody binds to the same epitope can be determined in various ways. In an exemplary method, the CHO cell expresses LGR5 on the cell membrane, or an alanine substitution mutant, preferably a mutant comprising one or more of substitutions M46A, F67A, R90A, or F91A. The test antibody was contacted with CHO cells and binding of the antibody to the cells was compared. The test antibody binds to the epitope if it binds to LGR5, and to a lesser extent to LGR5 with M46A, F67A, R90A, or F91A substitution. It is preferred to compare binding to a set of mutants each comprising a substitution of one alanine residue. Such binding studies are well known in the art. Typically the set comprises a single alanine substitution mutant covering substantially all amino acid residues. For LGR5, the group need only cover the extracellular portion of the protein, and certainly covers the portion that ensures association with the cell membrane when the cell is used. Expression of a particular mutant may be impaired, but this is readily detected by virtue of one or more LGR5 antibodies binding to a different region(s). If expression is also reduced for these control antibodies, the protein level or fold on the membrane is impaired for this particular mutant. The binding characteristics of the test antibody to this panel readily identify whether the test antibody exhibits reduced binding to mutants having M46A, F67A, R90A, or F91A substitutions, and thus whether the test antibody is an antibody of the invention. Reduced binding to mutants with M46A, F67A, R90A or F91A substitutions also identified an epitope within amino acid residues 21-118 of the sequence of figure 1. In preferred embodiments, the set comprises a D43A substitution mutant, a G44A substitution mutant, or both. Antibodies with the VH sequence of the VH of MF5816 exhibited reduced binding to these substitution mutants.

Without being bound by any theory, it is believed that amino acid residue I462 as depicted in figure 2; g465; k489; i491; n493; and C499 is involved in binding epitopes by antibodies comprising variable domains as indicated above. Participation in binding is preferably determined by observing reduced binding of the variable domain to EGFR with one or more of the amino acid residue substitutions selected from the group consisting of: I462A; G465A; K489A; I491A; N493A; and C499A.

In one aspect, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence depicted in figure 2. Preferably, the binding of the variable domain to EGFR is attenuated by means of one or more of the following amino acid residue substitutions in EGFR: I462A; G465A; K489A; I491A; N493A; and C499A. Binding of the antibody to human EGFR preferably interferes with binding of EGF to the receptor. The epitope on EGFR is preferably a conformational epitope. In one aspect, the epitope is located within amino acid residues 420-480 of the sequence depicted in FIG. 2, preferably 430-480 of the sequence depicted in FIG. 2; preferably within 438-469 of the sequence depicted in figure 2.

Without being bound by theory, it is believed that the contact residue of the epitope, i.e. the position at which the variable domain contacts human EGFR, may be I462; k489; i491; and N493. Amino acid residues G465 and C499 may be indirectly involved in binding of an antibody to EGFR.

The variable domain that binds human EGFR is preferably a variable domain having a heavy chain variable region comprising at least the CDR3 sequence of VH of MF3755 as depicted in figure 3, or a CDR3 sequence that differs from the CDR3 sequence of VH of MF3755 as depicted in figure 3 by at most three, preferably at most two, preferably no more than one amino acid.

The variable domain that binds human EGFR is preferably a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences of VH of MF3755 as depicted in figure 3; or CDR1, CDR2 and CDR3 sequences of VH of MF3755 as depicted in fig. 3 with at most three, preferably at most two, preferably at most one amino acid substitutions.

The variable domain that binds human EGFR is preferably a variable domain having a heavy chain variable region comprising the sequence of the VH chain of MF3755 as depicted in figure 3; or the amino acid sequence of the VH chain of MF3755 depicted in fig. 3, having up to 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain of MF3755.

In one embodiment, the present disclosure provides an antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5,

wherein the heavy chain variable region of the variable domain comprises at least one amino acid sequence selected from the group consisting of MF3370 as depicted in fig. 3; MF3755; a CDR3 sequence of an EGFR-specific heavy chain variable region of MF4280 or MF4289, or wherein the heavy chain variable region of the variable domain comprises a CDR sequence identical to a CDR sequence selected from the group consisting of MF3370 as depicted in fig. 3; MF3755; the CDR3 sequences of the VH of MF4280 or MF4289 differ in at most three, preferably at most two, preferably no more than one amino acid of the heavy chain CDR3 sequences. The variable domain preferably comprises a polypeptide comprising at least MF3370 as depicted in fig. 3; MF3755; the heavy chain variable region of the CDR3 sequence of MF4280 or MF 4289.

Said variable domain preferably comprises a polypeptide comprising at least one amino acid sequence selected from the group consisting of MF3370 as depicted in fig. 3; MF3755; a heavy chain variable region of CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region of MF4280 or MF4289, or comprising at least one amino acid sequence selected from the group consisting of MF3370 as depicted in fig. 3; MF3755; the heavy chain variable region of the EGFR-specific heavy chain variable region of the group consisting of MF4280 or MF4289 has CDR1, CDR2 and CDR3 sequences that differ in at most three, preferably at most two, preferably at most one amino acid. The variable domain preferably comprises a polypeptide comprising at least MF3370 as depicted in fig. 3; MF3755; the heavy chain variable region of CDR1, CDR2, and CDR3 sequences of MF4280 or MF 4289. The preferred heavy chain variable region is MF3755. Another preferred heavy chain variable region is MF4280.

An antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5, wherein the EGFR binding variable domain has a CDR3, CDR1, CDR2 and CDR3 and/or VH sequence as indicated above, preferably has a variable domain that binds LGR5, the variable domain comprising at least one amino acid selected from the group consisting of MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, or a CDR3 sequence of an LGR 5-specific heavy chain variable region selected from the group consisting of MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, differs in at most three, preferably at most two, preferably no more than one amino acid. The variable domain preferably comprises a polypeptide comprising at least MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or the heavy chain variable region of the CDR3 sequence of MF5818.

The LGR5 variable domain preferably comprises a heavy chain variable region comprising at least one amino acid selected from the group consisting of MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or the CDR1, CDR2 and CDR3 sequences of LGR 5-specific heavy chain variable region of the group consisting of MF5818, or with a CDR selected from the group consisting of MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or a group consisting of MF5818, wherein the CDR1, CDR2 and CDR3 sequences of the LGR 5-specific heavy chain variable region differ in at most three, preferably at most two, preferably at most one amino acid heavy chain CDR1, CDR2 and CDR3 sequences. The variable domain preferably comprises a domain comprising at least MF5790 as depicted in fig. 3; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or the heavy chain variable region of the CDR1, CDR2 and CDR3 sequences of MF5818. Preferred heavy chain variable region is MF5790; MF5803; MF5814; MF5816; MF5817; or MF5818. Particularly preferred heavy chain variable region is MF5790; MF5814; MF5816; and MF5818; preferred are MF5814, MF5818 and MF5816, and the heavy chain variable region MF5816 is particularly preferred. Another preferred heavy chain variable region is MF5818.

Antibodies comprising one or more variable domains having the heavy chain variable region MF3755 or one or more CDRs thereof have been shown to be of better effectiveness when used to inhibit growth of EGFR ligand responsive cancers or cells. In the case of bispecific or multispecific antibodies, the arms of an antibody comprising a variable domain having heavy chain variable region MF3755 or one or more CDRs thereof are in good combination with the arms comprising a variable domain having heavy chain variable region MF5818 or one or more CDRs thereof.

The VH chain of the variable domain that binds EGFR or LGR5 may have one or more amino acid substitutions relative to the sequence depicted in figure 3. The VH chain preferably has the amino acid sequence of EGFR or LGR5 VH of figure 3 with up to 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably with 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain sequence of figure 3.

The CDR sequences may have one or more amino acid residue substitutions relative to the CDR sequences in the figures. Such one or more substitutions are, for example, made for optimization purposes, preferably to improve the binding strength or stability of the antibody. Optimization is for example performed by means of a mutation-inducing procedure, wherein preferably after testing the stability and/or binding affinity of the resulting antibody, an improved EGFR-specific CDR sequence or an LGR 5-specific CDR sequence is preferably selected. The person skilled in the art is fully capable of generating antibody variants comprising at least one altered CDR sequence according to the invention. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue (e.g., isoleucine, valine, leucine or methionine) for another, and the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids or glutamines for asparagine.

Preferably, up to 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably 1, 2, 3,4 or 5 amino acid substitutions as mentioned in a VH or VL as specified herein are preferably conservative amino acid substitutions. Amino acid insertions, deletions and substitutions in a VH or VL as specified herein are preferably not present in a CDR3 region. The amino acid insertions, deletions and substitutions mentioned are preferably also not present in the CDR1 and CDR2 regions. The amino acid insertions, deletions and substitutions mentioned are preferably also not present in the FR4 region.

The up to 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably 1, 2, 3,4 or 5 amino acid substitutions mentioned are preferably conservative amino acid substitutions, insertions, deletions, substitutions or combinations thereof preferably not in the CDR3 region of the VH chain, preferably not in the CDR1, CDR2 or CDR3 region of the VH chain, and preferably not in the FR4 region.

An antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 preferably comprises

-the amino acid sequence of VH chain MF3755 as depicted in figure 3; or

-the amino acid sequence of VH chain MF3755 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and is provided with

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5790 as depicted in fig. 3; or

-the amino acid sequence of VH chain MF5790 as depicted in fig. 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

-the amino acid sequence of VH chain MF3755 as depicted in figure 3; or

-the amino acid sequence of VH chain MF3755 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to said VH; and is

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5803 as depicted in fig. 3; or

-the amino acid sequence of VH chain MF5803 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

-the amino acid sequence of VH chain MF3755 as depicted in figure 3; or

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5814 as depicted in figure 3; or

-the amino acid sequence of VH chain MF5814 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

-the amino acid sequence of VH chain MF3755 as depicted in figure 3; or

-the amino acid sequence of VH chain MF3755 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and is

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5816 as depicted in figure 3; or

-the amino acid sequence of VH chain MF5816 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

-the amino acid sequence of VH chain MF3755 as depicted in figure 3; or

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5817 as depicted in figure 3; or

-the amino acid sequence of VH chain MF5817 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

Amino acid sequence of VH chain MF3755 as depicted in FIG. 3 or

Wherein the VH chain of the variable domain that binds LGR5 comprises

-the amino acid sequence of VH chain MF5818 as depicted in figure 3; or

-the amino acid sequence of VH chain MF5818 as depicted in figure 3, having at most 15, preferably 1, 2, 3,4,5, 6, 7, 8, 9 or 10 and preferably having 1, 2, 3,4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

Additional variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries containing rearranged human IGKVl-39/IGKJl VL regions (De Kruif et al Biotechnol bioeng.2010 (106) 741-50), and collections of VH regions that incorporate amino acid substitutions into the amino acid sequences of EGFR or LGR5 VH regions disclosed herein, as previously described (e.g., WO 2017/069628). Phage encoding Fab regions that bind EGFR or LGR5 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

The light chain variable regions of the VH/VL EGFR and LGR5 variable domains of the EGFR/LGR5 antibody may be the same or different. In some embodiments, the VL region of the VH/VL EGFR variable domain of the EGFR/LGR5 antibody is similar to the VL region of the VH/VL LGR5 variable domain. In certain embodiments, the VL region in the first and second VH/VL variable domains is the same.

In certain embodiments, the light chain variable regions of one or both VH/VL variable domains of an EGFR/LGR5 antibody comprise a common light chain variable region. In some embodiments, the common light chain variable region of one or both VH/VL variable domains comprises a germline IgV kappa 1-39 variable region V segment. In one embodiment, the light chain variable region of one or both VH/VL variable domains comprises a kappa light chain V segment igvk 1-39 x 01.IgV kappa 1-39 is a shorthand for immunoglobulin variable kappa 1-39 genes. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39. Gene external Id is HGNC:5740; entrez gene: 28930; ensembl: ENGG 00000242371. Suitable amino acid sequences for the V regions are provided in figure 4. The V region may be combined with one of the five J regions. Preferably the J regions are jk1 and jk5 and the joining sequences are designated IGKV1-39/jk1 and IGKV1-39/jk5; the alternative names IgV κ 1-39 × 01/IGJ κ 1 × 01 or IgV κ 1-39 × 01/IGJ κ 5 × 01 (named according to IMGT database global information network IMGT. In certain embodiments, the light chain variable region of one or both VH/VL variable domains comprises a kappa light chain IgV κ 1-39 × 01/IGJ κ 1 × 01 or IgV κ 1-39 × 01/IGJ κ 1 × 05 (depicted in fig. 4).

In some embodiments, the light chain variable region of one or both VH/VL variable domains of an EGFR/LGR5 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (depicted in figure 4), an LCDR2 comprising the amino acid sequence AAS (depicted in figure 4), and an LCDR3 comprising the amino acid sequence QQSYSTP (depicted in figure 4) (i.e., the CDRs of IGKV1-39 according to IMGT). In some embodiments, the light chain variable region of one or both VH/VL variable domains of an EGFR/LGR5 antibody comprises LCDR1 comprising the amino acid sequence QSISSY (depicted in figure 4), LCDR2 comprising the amino acid sequence AASLQS (depicted in figure 4), and LCDR3 comprising the amino acid sequence QQSYSTP (depicted in figure 4).

In some embodiments, one or both VH/VL variable domains of an EGFR/LGR5 antibody comprises a light chain variable region comprising an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence set forth in figure 4. In some embodiments, one or both VH/VL variable domains of an EGFR/LGR5 antibody comprises a light chain variable region comprising an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical or 100% identical to the amino acid sequence set forth in figure 4.

For example, in some embodiments, the variable light chain of one or both VH/VL variable domains of an EGFR/LGR5 antibody may have 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof relative to the sequence in figure 4. In some embodiments, the light chain variable region of one or both VH/VL variable domains of an EGFR/LGR5 antibody comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1 and preferably 0 amino acid insertion, deletion, substitution, addition or combinations thereof relative to a specified amino acid sequence.

In other embodiments, the light chain variable region of one or both VH/VL variable domains of an EGFR/LGR5 antibody comprises an amino acid sequence as the sequence depicted in figure 4. In certain embodiments, the two VH/VL variable domains of the EGFR/LGR5 antibody comprise the same VL region. In one embodiment, the VL of both VH/VL variable domains of an EGFR/LGR5 bispecific antibody comprises the amino acid sequence set forth in figure 4. In one embodiment, the VL of both VH/VL variable domains of an EGFR/LGR5 bispecific antibody comprises the amino acid sequence set forth in figure 4.

The EGFR/LGR5 antibody as described herein is preferably a bispecific antibody having two variable domains, one variable domain binding to EGFR and the other variable domain binding to LGR5 as described herein. EGFR/LGR5 bispecific antibodies for use in the methods disclosed herein may be provided in a variety of forms. Many different forms of bispecific antibodies are known in the art and have been reviewed by Kontermann (Drug discovery Today,2015, 7 months; 20 (7): 838-47 MAbs,2012 3 months-4 months; 4 (2): 182-97) and Spiess et al, (Alternative molecular formats and therapeutic applications for biological antibodies. Mol. Immunol. 2015. Http:// dx. Doi. Org/10.1016/j. Molimem. 2015. 01.003), each of which is incorporated herein by reference. For example, a bispecific antibody format that is not typical of antibodies having two VH/VL combinations has at least one variable domain comprising a heavy chain variable region and a light chain variable region. This variable domain can be linked to single chain Fv fragments, monofunctional antibodies (monobodies), VH and Fab fragments that provide secondary binding activity.

In some embodiments, the EGFR/LGR5 bispecific antibody used in the methods provided herein is generally of the human IgG subclass (e.g., igG1, igG2, igG3, igG 4). In certain embodiments, the antibody is a human IgG1 subclass. Full-length IgG antibodies are preferred due to their favorable half-life and for reasons of low immunogenicity. Thus, in certain embodiments, the EGFR/LGR5 bispecific antibody is a full length IgG molecule. In one embodiment, the EGFR/LGR5 bispecific antibody is a full length IgG1 molecule.

Thus, in certain embodiments, an EGFR/LGR5 bispecific antibody comprises a crystallizable fragment (Fc). The Fc of EGFR/LGR5 bispecific antibodies is preferably composed of human constant regions. The constant region or Fc of the EGFR/LGR5 bispecific antibody may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the naturally occurring human antibody constant region. For example, in certain embodiments, the Fab arm of a bispecific antibody can further comprise a modified Fc region comprising a modification that facilitates formation of the bispecific antibody, facilitates stability, and/or other features described herein.

Bispecific antibodies are typically produced by cells expressing the nucleic acid(s) encoding the antibody. Thus, in some embodiments, the bispecific EGFR/LGR5 antibodies disclosed herein are produced by providing a cell comprising one or more nucleic acids encoding the heavy and light chain variable and constant regions of the bispecific EGFR/LGR5 antibody. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, most preferably a human cell. Suitable cells are any cells capable of containing and preferably capable of producing an EGFR/LGR5 bispecific antibody.

Cells suitable for antibody production are known in the art and include hybridoma cells, chinese Hamster Ovary (CHO) cells, NS0 cells, or PER-C6 cells. Cell lines for large-scale production of antibodies have been developed by various institutions and companies, for example, for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells or per.c6 cells. In a particularly preferred embodiment, the cell is a human cell. Preferably, the cell is transformed with an adenovirus E1 region or a functional equivalent thereof. C6 cell line or its equivalent is a preferred example of such a cell line. In a particularly preferred embodiment, the cell is a CHO cell or variant thereof. Preferably, the variant utilizes a Glutamine Synthetase (GS) vector system for expression of the antibody. In a preferred embodiment, the cell is a CHO cell.

In some embodiments, the cells express different light and heavy chains that make up the EGFR/LGR5 bispecific antibody. In certain embodiments, the cell expresses two different heavy chains and at least one light chain. In a preferred embodiment, the cells express a "common light chain" as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, using methods known in the art for the production of bispecific IgG (WO 2013/157954; incorporated herein by reference), the respective VH regions were cloned into expression vectors with a rearranged human IGKV1 39/IGKJ1 (huV κ 1) light chain, previously shown to be capable of pairing with more than one heavy chain, thereby producing antibodies with diverse specificities, which facilitate the production of bispecific molecules (De Kruif et al j.mol.biol.2009 (387) 548 58.

Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce each of 50% bispecific antibodies and 25% monospecific antibodies (i.e., with identical heavy-light chain combinations). Several methods have been disclosed to prioritize the generation of bispecific antibodies over the generation of individual monospecific antibodies. This is typically achieved by modifying the constant region of the heavy chain such that it favors heterodimerization (i.e., heavy chain dimerization with another heavy/light chain combination) over homodimerization. In a preferred embodiment, the bispecific antibodies of the invention comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. This technology describes various ways in which such heterodimerization of heavy chains can be achieved.

A preferred method for producing an EGFR/LGR5 bispecific antibody is disclosed in US 9,248,181 and US 9,358,286. In particular, preferred mutations that produce essentially only bispecific full length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) ("KK variant" heavy chain) in the first CH3 domain and the amino acid substitutions L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. As described previously, DE and KK variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains (DE homodimers) or of KK variant heavy chains (kkkkkkkk homodimers) hardly occurs due to the strong repulsion between charged residues in the inter CH3-CH3 interfaces of identical heavy chains.

Thus, in one embodiment, a heavy chain/light chain combination comprising a variable domain that binds EGFR comprises a DE variant of the heavy chain. In this embodiment, a heavy chain/light chain combination comprising a variable domain that binds LGR5 comprises a KK variant of a heavy chain.

The binding of the candidate EGFR/LGR5 IgG bispecific antibody can be tested using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be assessed by flow cytometry (according to FACS procedure as previously described in WO 2017/069628). In one embodiment, binding of the candidate EGFR/LGR5 bispecific antibody to LGR5 on CHO cells is demonstrated by means of flow cytometry according to standard procedures known in the art. Binding to CHO cells was compared to CHO cells that have not been transfected with expression cassettes for EGFR and/or LGR5. Determining binding of the candidate bispecific IgG1 to EGFR using CHO cells transfected with EGFR expression constructs; LGR5 monospecific antibodies and EGFR monospecific antibodies, as well as irrelevant IgG1 isotype control mabs were included in the assay as controls (e.g., antibodies that bind LGR5 and another antigen such as Tetanus Toxin (TT)).

The affinity of LGR5 and EGFR Fab of candidate EGFR/LGR5 bispecific antibodies to their targets can be measured by Surface Plasmon Resonance (SPR) techniques using BIAcore T100. Briefly, anti-human IgG mouse monoclonal antibodies (Becton and Dickinson, catalog No. 555784) were coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS/EDC). Subsequently, bsAb was captured onto the sensor surface. Subsequently, the recombinant purified antigens human EGFR (nano Biological Inc, catalog number 11896-H07H) and human LGR5 protein were flowed over the sensor surface at a range of concentrations and association and dissociation rates were measured. After each cycle, the sensor surface was regenerated by means of HCl pulses and bsAb was captured again. From the obtained sensor profiles, association and dissociation rates and affinity values for binding to human LGR5 and EGFR were determined using BIAevaluation software, as previously described for CD3 in US 2016/0368988.

The antibodies as disclosed herein are typically bispecific full length antibodies, preferably of the human IgG subclass, preferably of the human IgG1 subclass. Such antibodies have good ADCC properties that can be enhanced, if necessary, by techniques known in the art, have a favorable half-life when administered in vivo to humans, and CH3 engineering techniques exist that can provide modified heavy chains that form heterodimers in preference to homodimers when co-expressed in clonal cells.

When the antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by modifying the antibody constant region. Another way to improve ADCC activity of an antibody is to interfere with glycosylation pathways by means of enzymes, resulting in reduced fucose. There are several in vitro methods for determining the efficacy of an antibody or effector cell in eliciting ADCC. Among them, there are a chromium (51), [ Cr51] release assay, a europium [ Eu ] release assay and a sulfur-35 [ S35] release assay. Typically, a labeled target cell line expressing a certain surface-exposed antigen is incubated with an antibody specific for the antigen. After washing, effector cells expressing the Fc receptor CD16 were co-incubated with antibody-labeled target cells. Target cell lysis is then measured from the release of intracellular markers by scintillation counting or spectrophotometry.

Bispecific antibodies as disclosed herein can be enhanced by ADCC. In one embodiment, the bispecific antibody can be non-fucose modified. The bispecific antibody preferably comprises a reduced amount of fucosylation of the N-linked carbohydrate structure in the Fc region when compared to the same antibody produced in normal CHO cells.

Antibodies comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 can further comprise one or more additional variable domains that can bind one or more other targets. Other targets are preferably proteins, preferably membrane proteins comprising extracellular portions. Membrane proteins as used herein are cell membrane proteins, e.g. proteins in the outer membrane of a cell, i.e. the membrane separating the cell from the outside world. The membrane protein has an extracellular portion. A membrane protein is at least on a cell if it contains a transmembrane region in the cell membrane of the cell.

Antibodies with more than two variable domains are known in the art. For example, it is possible to link additional variable domains to the constant portion of the antibody. Antibodies with three or more variable domains are preferably multivalent multimeric antibodies as described in PCT/NL2019/050199, which is incorporated herein by reference.

In one embodiment, the antibody is a bispecific antibody comprising two variable domains, wherein one variable domain binds the extracellular portion of EGFR and the other variable domain binds the extracellular portion of LGR5. The variable domain is preferably a variable domain as described herein.

The functional portion of the antibody as described herein comprises at least a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 as described herein. Thus, it comprises the antigen binding portion of an antibody as described herein, and typically contains the variable domains of an antibody. The variable domains of the functional parts may be single chain Fv fragments or so-called single domain antibody fragments. Single domain antibody fragments (sdabs) are antibody fragments that have a single monomeric variable antibody domain. Like intact antibodies, they are capable of binding selectively to specificityAn antigen. The single domain antibody fragments have molecular weights of only 12-15kDa, are much smaller than the common antibody consisting of two heavy protein chains and two light chains (150-160 kDa), and are even smaller than Fab fragments (about 50kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25kDa, two variable domains, one from the light chain and one from the heavy chain). Single domain antibodies are not themselves much smaller than normal antibodies (typically 90-100 kDa). Single domain antibody fragments are mainly engineered from heavy chain antibodies found in camelids; these fragments are referred to as VHH fragments

Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin neo-antigen receptor"), from which single domain antibody fragments, referred to as VNAR fragments, are obtained. An alternative approach splits the dimeric variable domain of a common immunoglobulin G (IgG) from human or mouse into monomers. While most current research on single domain antibodies is based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to epitopes of interest. Non-limiting examples of such variable domains of antibody portions are VHH, human domain antibodies (dAb) and single antibodies (Unibody). Preferably the antibody part or derivative has at least two variable domains of the antibody or an equivalent thereof. Non-limiting examples of such variable domains or their equivalents are F (ab) fragments and single chain Fv fragments. The functional portion of the bispecific antibody comprises an antigen-binding portion, or a derivative and/or analog of a binding portion, of the bispecific antibody. As mentioned above, the binding portion of an antibody is encompassed in the variable domain.

Also provided are antibodies, or functional portions, derivatives and/or analogs thereof (i.e., therapeutic compounds) as disclosed herein, and pharmaceutically acceptable carriers. Such pharmaceutical compositions are useful for the treatment of cancer, particularly gastric, esophageal or gastro-esophageal junction cancer. As used herein, the term "pharmaceutically acceptable" means approved by a governmental regulatory agency or listed in the U.S. pharmacopoeia (u.s.pharmacopoeia) or another generally recognized pharmacopoeia for use in animals, particularly humans, and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol ricinoleate and the like. Aqueous or physiological saline solutions and aqueous dextrose and glycerol solutions can be employed as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration may be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, and the like), the active compound may be encapsulated in a substance that protects the compound from the effects of acids and other natural conditions that may inactivate the compound.

Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example in a liquid carrier or adapted to be reconstituted into a liquid solution or suspension for intravenous administration. The compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage. Also included are solid formulations intended to be converted, immediately prior to use, to liquid formulations for oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions.

The disclosed therapeutic compounds can be administered according to a suitable dosage and by a suitable route (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In one embodiment, the subject is administered a single dose of an antibody or functional part, derivative and/or analogue thereof as disclosed herein. In some embodiments, the therapeutic compound will be administered repeatedly over the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3,4,5, 6, 7, 8, 9, 10, or more) doses of a therapeutic compound are administered to a subject in need of treatment. In some embodiments, the administration of the therapeutic compound can be performed weekly, biweekly, or monthly.

The clinician may utilize preferred dosages appropriate for the patient condition being treated. The dosage may depend on a variety of factors, including the stage of the disease, etc. It is within the skill of the artisan to determine the particular dose that should be administered based on the presence of one or more such factors. In general, treatment begins with a smaller dose than the optimal dose of the compound. Thereafter, the dosage is increased by a small amount until the optimum effect under these circumstances is reached. For convenience, the total daily dose may be divided and administered in portions during the day, if necessary. Intermittent therapy (e.g., one of three weeks or three of four weeks) may also be used.

In certain embodiments, the therapeutic compound is administered at a dose of 0.1, 0.3, 1, 2, 3,4,5, 6, 7, 8, 9, or 10 mg/kg body weight. In another embodiment, the therapeutic compound is administered at a dose of 0.5, 1, 2, 3,4,5, 6, 7, 8, 9, or 10 mg/kg body weight.

In a preferred embodiment, the therapeutic compound (i.e., an antibody or functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR 5) is provided to the subject at a dose of 1500 mg. Fixed doses offer a number of advantages over topical or body weight administration, as they shorten preparation times and reduce potential dose calculation errors. In some embodiments, the therapeutic compound is provided at a dose of at least 1100mg, preferably at a dose between 1100 and 2000mg, more preferably at a dose between 1100 and 1800 mg. As will be appreciated by those skilled in the art, the dosage may be administered over time. For example, the dose may be administered by IV, e.g. by infusion over 1-6 hours, preferably 2-4 hours. In some embodiments, the therapeutic compound is administered once every 2 weeks. In some embodiments, the fixed dose disclosed herein is suitable for adults and/or subjects having a body weight of at least 35 kg. Preferably, the subject suffers from gastric cancer, esophageal cancer or carcinoma of the gastro-esophageal junction.

In some embodiments, a pre-medication (pre-medication) regimen may be used. Such protocols may be useful for reducing the likelihood or severity of infusion-related reactions. Generally, steroids such as dexamethasone (dexchlorpheniramine) and/or antihistamines such as dexchlorpheniramine, diphenhydramine or chlorpheniramine are administered (e.g. orally, intravenously) prior to antibody therapy.

The treatment methods described herein generally continue as long as the clinician supervising the patient care deems the treatment method effective, i.e., the patient is responsive to the treatment. Non-limiting parameters indicative of a treatment method being effective may include one or more of: a decrease in tumor cells; inhibition of tumor cell proliferation; elimination of tumor cells; progression-free survival; suitable for appropriate reaction of tumor markers (if applicable).

With respect to the frequency of administration of the therapeutic compound, one of ordinary skill in the art will be able to determine the appropriate frequency. For example, a clinician may decide to administer a therapeutic compound relatively infrequently (e.g., once every two weeks), and gradually shorten the time period between doses tolerated by a patient. Exemplary lengths of time associated with a course of therapy according to the claimed method include: about one week; two weeks; about three weeks; about four weeks; about five weeks; about six weeks; about seven weeks; about eight weeks; about nine weeks; about ten weeks; about eleven weeks; about twelve weeks; about thirteen weeks; about fourteen weeks; about fifteen weeks; about sixteen weeks; about seventeen weeks; about eighteen weeks; about nineteen weeks; about twenty weeks; about twenty one weeks; about twenty-two weeks; about twenty-three weeks; about twenty-four weeks; about seven months; about eight months; about nine months; about ten months; about eleven months; about twelve months; about thirteen months; about fourteen months; about fifteen months; about sixteen months; about seventeen months; about eighteen months; about nineteen months; about twenty months; about twenty-one months; about twenty-two months; about twenty-three months; about twenty-four months; about thirty months; about three years; about four years; about five years; permanent (e.g., persistent maintenance therapy). The aforementioned duration may be associated with one or more cycles/cycles of treatment.

Any suitable means may be used to assess the efficacy of the treatment methods provided herein. In one embodiment, the reduction in the number of cancer cells is used as an objective response criterion to analyze the clinical efficacy of the treatment. Patients (e.g., humans) treated according to the methods disclosed herein preferably experience an improvement in at least one cancer condition. In some embodiments, one or more of the following may occur: the number of cancer cells can be reduced; prevention or delay of cancer recurrence; one or more symptoms associated with cancer may be alleviated to some extent. In addition, in vitro assays to determine T cell mediated lysis of target cells. In some embodiments, tumor assessment is based on CT scans and/or MRI scans, see, e.g., RECIST 1.1 guidelines (Criteria for Solid tumor Response assessment (Response Evaluation Criteria) in Solid tumors) (Eisenhauer et al, 2009Eur J Cancer 45: 228-247). Such assessments are typically made every 4-8 weeks after treatment.

In some embodiments, tumor cells are no longer detectable after treatment as described herein. In some embodiments, the subject is in partial or complete remission. In certain embodiments, the overall survival, median survival, and/or progression-free survival of the subject is increased.

The therapeutic compounds (i.e., comprising an antibody or functional portion, derivative and/or analog thereof that binds the variable domain of the extracellular portion of EGFR and binds the variable domain of the extracellular portion of LGR 5) may also be used with other well-known therapies (e.g., chemotherapy or radiation therapy) selected for their particular usefulness against the cancer being treated.

Methods for safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, montvale, N.J.07645-1742, USA); the disclosure of which is incorporated herein by reference.

It will be apparent to those skilled in the art that the administration of chemotherapeutic agent(s) and/or radiation therapy may vary depending on the disease being treated and the known effects of chemotherapeutic agent(s) and/or radiation therapy on the disease. Furthermore, according to the knowledge of a skilled clinician, the treatment regimen (e.g., dosage and time of administration) may vary in view of the observed effect of the administered therapeutic on the patient and in view of the observed response of the disease to the administered therapeutic.

The compounds and compositions disclosed herein are useful as therapeutics and for therapeutic treatment, and thus may be useful as medicaments and for methods of preparing medicaments.

All documents and references, including Genbank entries, patents and published patent applications and websites, described herein are each expressly incorporated by reference herein to the same extent as if written in whole or in part in this document.

For purposes of clarity and brevity, the features are described herein as part of the same or separate embodiments, however, it is to be understood that the scope of the invention may include embodiments having combinations of all or some of the described features.

The invention will now be described with reference to the following examples, which are illustrative only and not intended to be limiting of the invention. While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Examples

As used herein, "MFXXXX" wherein X is independently a number from 0 to 9 refers to a Fab comprising a variable domain, wherein VH has an amino acid sequence identified by the arabic number at position 4 depicted in figure 3. Unless otherwise indicated, the light chain variable regions of the variable domains typically have the sequence of fig. 4 b. The light chain in the examples has the sequence as depicted in fig. 4 a. "MFXXXXX VH" refers to the amino acid sequence of VH identified by the Arabic number at position 4. The MF further comprises a constant region of the light chain and a constant region of the heavy chain that typically interacts with the constant region of the light chain. The VH/variable regions of the heavy chains differ, and typically the CH3 regions also differ, with the CH3 domain of one of the heavy chains having a KK mutation and the CH3 domain of the other having a complementary DE mutation (see PCT/NL2013/050294 (published as WO 2013/157954) and fig. 5d and 5e for reference the bispecific antibody in the embodiments has an Fc tail with a KK/DE CH3 heterodimerization domain as indicated in fig. 5, a CH2 domain and a CH1 domain, a common light chain as indicated in fig. 4a and a VH as specified by the MF number, for example, the bispecific antibody indicated by MF3755 x MF5816 has the above general sequence, and a variable domain with a VH having a sequence of MF3755 and a variable domain with a VH having a sequence of MF 5816.

The amino acid and nucleic acid sequences of the various heavy chain variable regions (VH) are indicated in fig. 3. The bispecific antibody EGFR/LGR5, MF3755 × MF5816; the heavy chain variable regions MF3755 and MF5816 and the common light chain are comprised and include modifications to enhance ADCC by non-fucose modifications, as well as other LGR5 and EGFR combinations as depicted in fig. 3 have been shown to be effective in WO 2017/069628.

Production of bispecific antibodies

Bispecific antibodies were generated by transient co-transfection with two plastids encoding iggs with different VH domains using dedicated CH3 engineering techniques to ensure efficient heterodimerization and bispecific antibody formation. The common light chain is also co-transfected into the same cell, either on the same plastid or on another plastid. In our applications (e.g., WO2013/157954 and WO2013/157953, incorporated herein by reference), we have disclosed methods and means for generating bispecific antibodies from single cells, wherein means are provided to prioritize bispecific antibody formation over monospecific antibody formation. These methods may also be advantageously used in the present invention. In particular, preferred mutations that generate essentially only bispecific full length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, such as L351K and T366K (numbering according to EU numbering) ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, such as L351D and L368E ("DE variant" heavy chain), or vice versa (see fig. 5D and 5E). It was previously demonstrated in the mentioned application that negatively charged DE variant heavy chains and positively charged KK variant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE variant heavy chain (DE-DE homodimer) or of the KK variant heavy chain (KK-KK homodimer) hardly occurs due to the strong repulsion between charged residues in the inter-CH 3 interfaces of identical heavy chains.

The VH gene described above that binds the variable domain of LGR5 was cloned into a vector encoding a positively charged CH3 domain. VH genes that bind the variable domains of EGFR, such as those disclosed in WO2015/130172 (incorporated herein by reference), are cloned into vectors encoding negatively charged CH3 domains. Adapted 293F Freestyle cells were grown in suspension in T125 flasks on a shaker deck to a density of 3.0 × 10e6 cells/ml. Cells were seeded at a density of 0.3-0.5 × 10e6 viable cells/ml in each well of a 24-well plate. Cells were transiently transfected with a mixture of two plastids, encoding different antibodies, cloned into a proprietary vector system. Seven days after transfection, cell supernatants were collected and filtered through 0.22 μ M filters (Sartorius). The sterile supernatant was stored at 4 ℃ until antibody purification.

IgG purification and quantification

Purification was performed using protein a affinity chromatography in a filter tray under sterile conditions. First, the pH of the medium was adjusted to pH 8.0, and then the IgG containing supernatant was incubated with protein A Sepharose CL-4B beads (50% v/v) (Pierce) for 2 hours at 25 ℃ at 600rpm on a shaking platform. Next, the beads were collected by filtration. Beads were washed twice with PBS pH 7.4. Subsequently, bound IgG was eluted with 0.1M citrate buffer at pH 3.0, and the eluate was subsequently neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using multiscreen Ultracel 10 multi-disc (Millipore). Finally, samples were collected in PBS pH 7.4. IgG concentrations were measured using Octet. The protein samples were stored at 4 ℃.

To determine the amount of purified IgG, the antibody concentration was determined by Octet analysis using a protein a biosensor (Forte-Bio, according to supplier's recommendations) using whole human IgG (Sigma Aldrich, catalog No. I4506) as standard.

The following bispecific antibodies are suitable for use in this example and in the methods of the invention: MF 3370X MF5790, MF 3370X 5803, MF 3370X 5805, MF 3370X 5808, MF 3370X 5809, MF 3370X 5814, MF 3370X 5816, MF 3370X 5817, MF 3370X 5818, MF 3755X MF5790, MF 3755X 5803, MF 3755X 5805, MF 3755X 5808, MF 3755X 5809, MF 3755X 5814, MF 3755X 5816, MF 3755X 5817, MF 3755X 5818, MF 3755X MF4280 × MF5790, MF4280 × 5803, MF4280 × 5805, MF4280 × 5808, MF4280 × 5809, MF4280 × 5814, MF4280 × 5816, MF4280 × 5817, MF4280 × 5818, MF4289 × MF5790, MF4289 × 5803, MF4289 × 5805, MF4289 × 5808, MF4289 × 5809, MF4289 × 5814, MF4289 × 5816, MF4289 × 5817 and MF4289 × 5818. Each bispecific antibody comprises two VH designated by MF number capable of binding to EGFR and LGR5, respectively, further comprising a VH having a sequence as set forth by SEQ ID NO:136 (FIG. 5 d) and SEQ ID NO:138 (fig. 5 e) the Fc tail of the KK/DE CH3 heterodimerization domain, as indicated by SEQ ID NO:134 (fig. 5 c) and a CH2 domain as indicated by SEQ ID NO:131 (fig. 5 a) a CH1 domain as indicated by SEQ ID NO:121 (FIG. 4) common light chain.

Example 1: evaluation of anti-EGFR x anti-LGR 5 candidates against various cancers:

mouse model selection

A series of patient-derived xenograft (PDX) models derived from surgically resected human primary tumors have been developed by crow Biosciences inc (crow Bioscience database, http:// hub. Crownbio. Com). The PDX model is clinically and molecularly labeled and faithfully represents the clinical epidemiology of the respective tumor. These models can be injected subcutaneously in the flanks of immunodeficient mice. EGFR and LGR5 expression was tested for different cancer models, as analyzed by RNA sequencing (RNAseq) (see table 1). A panel of PDX models found to exhibit high EGFR and LGR5 expression levels of esophageal and gastric cancer were selected to test the efficacy of MF3755 x MF5816 bispecific antibodies. For esophageal cancer, 4 PDX models were selected, and for gastric cancer, 8 PDX models were selected. Details of the PDX model, including the cancer subtype, known driver mutations, EGFR/LGR5 expression and time to randomization are described in (table 1).

TABLE 1

Features derived from PDX models of esophageal and gastric cancer patients.

LGR5 and EGFR expression were determined by RNA sequencing (RNAseq). The mutational status of oncogenic drivers was determined by means of genotypic analysis. Also mentioned is the achievement of randomization (mean tumor achievement of approximately 100-150 mm) ³ Time), and growth characteristics of the tumor. ESCC = esophageal squamous cell carcinoma; EAC = esophageal adenocarcinoma; ADC = adenocarcinoma; n/a = unavailable or not applicable.

Method

Fresh tumor tissue for inoculation was collected from mice bearing established primary human tumors. Depending on the model, tumor fragments (2-3 mm in diameter) were inoculated subcutaneously on the upper right dorsal side of 6-8 week old female BALB/c nude or NOD/SCID mice. When tumors reached 100-150mm3 size, mice were randomly grouped. A total of 6 mice, 3 control mice and 3 mice treated with MF3755 × MF5816 bispecific antibody were enrolled for each model. For 6 weeks, mice received 0.5mg bispecific antibody intraperitoneally in 200 μ l injection volume per week (approximately 25 mg/kg/week), regardless of their body weight. Control mice received PBS (200. Mu.l). After a 6-week treatment period, tumor growth was monitored for an additional 3 weeks. If the mice reached the humane endpoint, they were sacrificed earlier than day 63.

As a result:

in the 8 gastric PDX models tested, MF3755 × MF5816 bispecific antibody treatment significantly reduced tumor growth in 6 models (fig. 6 a). Of these 8 models, 3 (GA 0429, GA6833 and GA 6891) showed lower tumor volumes at the end of the observation period than at the beginning of the treatment, indicating a strong tumor suppression effect of the bispecific antibody in gastric cancer. Model GA2434 gives some responses. With respect to the esophageal PDX model, all four test models responded to bispecific antibody treatment, with model ES2356 responding most strongly (fig. 6 b). The results obtained with the MF3755 × MF5816 bispecific antibody of this example showing statistically significant (p < 0.0001) treatment efficacy were obtained in the case of model ES 11065.

Example 2: dose amplification of anti-EGFR x anti-LGR 5 antibodies against patients with EAC, GAC and GEJAC and the efficacy is as follows:

phase 1 dose escalation study in advanced solid tumors

Design of research

A phase 1 open-label multicenter study was performed with an initial dose escalation section to determine the recommended phase 2 dose (RP 2D) of anti-EGFR x anti-LGR 5 bispecific antibody against solid tumors in mCRC patients, starting at a 5mg fixed dose. Once RP2D was established, antibodies were further evaluated in the amplification part of the study, including in patients diagnosed with EAC, GAC and GEJAC. Antibody safety, PK, immunogenicity, and primary anti-tumor activity were characterized in all patients, and biomarker analysis was performed, including EGFR and LGR5 status.

Dose escalation

In the dose escalation section, patients with metastatic colorectal cancer (mCRC) adenocarcinoma who were previously treated in a metastatic context with standard approved therapies including oxaliplatin (oxaliplatin), irinotecan, and fluoropyrimidine (5-FU and/or capecitabine), with or without an anti-angiogenic agent, and anti-EGFR against KRAS and NRAS wild-type RASwt, are treated.

PK models were generated based on available bispecific antibody serum concentration data from preliminary and GLP cynomolgus monkey toxicology studies. After allometric scaling (allometric scaling), this model was used to predict antibody exposure in humans. The initial dose of antibody was 5mg (fixed dose) IV every 2 weeks with a 4-week cycle. Up to 11 dose levels will be studied: 5. 20, 50, 90, 150, 225, 335, 500, 750, 1100 and 1500mg (fixed dose). The dose amount, dose increment, and dosing frequency for each patient and each group were subject to variation based on patient safety, PK and PD data, however, the dose would not exceed 4500mg per cycle.

Dose Limiting Toxicity (DLT)

Any of the following clinical toxicities and/or laboratory abnormalities that occurred during the first cycle (28 days) and were considered by the investigator to be associated with antibody treatment would be considered DLT:

hematological toxicity:

grade 4 neutropenia (absolute neutrophil count [ ANC ] < 0.5X 109 cells/liter), lasting ≥ 7 days

Grade-3-4 febrile neutropenia

Grade 4 thrombocytopenia

-thrombocytopenia grade 3 associated with bleeding episodes

Other grade 4 hematological toxicity

Grade 3-4 non-hematological AE and laboratory toxicity, except:

-3-4 grade infusion related reactions

Grade 3 skin toxicity with recovery to grade 2 within 2 weeks under optimal treatment

Grade 3 diarrhea, nausea and/or vomiting to grade ≦ 1 or baseline within 3 days with optimal treatment

Grade 3 electrolyte abnormalities that regress within 48 hours under optimal treatment

Grade 3-4 liver abnormalities lasting ≦ 48 hours

Any liver function abnormality that meets the definition of Hai's Law.

Any drug related toxicity that persists for more than 15 days preventing the next second dose.

Dose amplification

In the amplification part, bispecific antibodies will be administered with RP2D in patients with EAC, GAC or GEJAC. Once RP2D has been determined, additional patients will be treated with this dose and schedule to further characterize the safety, tolerability, PK and immunogenicity of the antibodies, and to conduct preliminary assessments of anti-tumor activity and biomarker assessment. The malignant disease treated will be known to co-express both targets (i.e., LGR5 and EGFR) and may have previous signs of sensitivity to EGFR inhibition.

Antibody treatment in patients with EAC, GAC or GEJAC will be explored, e.g. 10 to 20 patients for each indication, possibly expanded to 40 patients, subject to signs of primary anti-tumor activity). The Safety of RP2D will be continuously assessed by the Safety Monitoring Committee (Safety Monitoring Committee) during the amplification part of the study. If for any set, the DLT incidence exceeds a predefined threshold of 33%, enrollment for that set will be suspended and a full review of the security, PK and biomarkers will be made by the SMC in order to determine if it is safe to continue accumulating in that set. At that time the overall safety of the drug will also be questioned.

Investigative therapy and regimens

anti-EGFR x anti-LGR 5 bispecific antibodies were formulated as clear solutions for IV infusion. IV infusions were performed every 2 weeks using standard infusion procedures with a starting dose of 5mg (fixed dose) and a recommended phase 2 dose of 1500mg (fixed dose). Once RP2D was reached, dose escalation was discontinued. During cycle 1, infusions must be administered over a minimum of 4 hours. Subsequent infusions following cycle 1 can be shortened to 2 hours at the discretion of the investigator and in the absence of IRR.

The cycle was considered to be 4 weeks. For each patient, a6 hour observation period was performed after the infusion of the initial antibody infusion was initiated, the second infusion was performed for a4 hour observation period, and all subsequent administrations were performed for a minimum 2 hour observation period, corresponding to at least the infusion duration. The antibody is administered every 2 weeks at 2 to 4 hour IV infusion for a period of 4 weeks. Day 1 of the subsequent cycle is at day 29, or after recovery from any adverse effects associated with the previous cycle.

Duration of treatment

Study treatment was administered until progressive disease was confirmed (according to RECIST 1.1), unacceptable toxicity, consent was withdrawn, patient noncompliance, investigator decision (e.g., clinical worsening) or antibody discontinuation for > 6 consecutive weeks. After the last antibody infusion, patients were followed for at least 30 days for safety and until all relevant toxicities were restored or stabilized, and patients were followed for 12 months for disease progression and survival status.

Efficacy assessment

Every 8 weeks after treatment initiation, tumor assessments were based on CT/MRI according to RECIST 1.1 comparisons (Eisenhauer et al, 2009Eur J Cancer 45. Objective responses must be confirmed at least 4 weeks after the first observation. Patients with bone metastases at baseline or with suspected lesions in the study were scanned for bone as indicated clinically. Circulating blood tumor markers, including carcinoembryonic antigen (CEA), were evaluated at the time of screening and day 1 of each cycle.

Claims

1. An antibody or functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in treating cancer in a subject, wherein said use comprises providing the subject with a fixed dose of 1500mg of the antibody or functional part, derivative and/or analogue thereof.

2. An antibody or a functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in treating gastric, esophageal or gastro-esophageal junction cancer in a subject.

3. An antibody or a functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 for use in the treatment of gastric, esophageal or gastro-esophageal junction cancer in a Her 2-negative individual.

4. An antibody or functional part, derivative and/or analogue thereof for use according to claim 2 or 3, wherein said use comprises providing the subject with a fixed dose of 1500mg of said antibody or functional part, derivative and/or analogue thereof.

5. An antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein the antibody or functional part, derivative and/or analogue thereof is provided intravenously.

6. The antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein the cancer has a mutation in one or more genes selected from: TP53, MLH1, PIK3CA, CDKN2A, UGT1A8, BRAF, PTEN and KRAS, preferably wherein the cancer has a mutation in one or more genes selected from: TP53, MLH1, CDKN2A, UGT1A8, BRAF and PTEN.

7. An antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer has one or more mutations selected from: TP 53R 196T; TP 53R 342T; TP 53R 248Q; MLH 1V 384D; PIK3CA H1047R; CDKN2A W110T; UGT1A1G71R; UGT1 A8G 71R; and KRAS G12C.

8. The antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein the administration of said antibody or functional part, derivative and/or analogue thereof is weekly, biweekly or monthly.

9. The antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein the antibody or functional part, derivative and/or analogue thereof is administered once every 2 weeks.

10. The antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer has a mutation in a gene encoding TP53, preferably wherein the mutation is R196T.

11. The antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably wherein the mutation is V384D.

12. The antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer has a mutation in a gene encoding TP53, preferably wherein the mutation is R248Q; the cancer has a mutation in the gene encoding PIK3CA, preferably wherein the mutation is H1047R; the cancer has a mutation in a gene encoding CDKN2A, preferably wherein the mutation is W110T; the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in a gene encoding UGT1A8, preferably wherein the mutation is G71R.

13. An antibody or a functional part, derivative and/or analogue thereof for use according to any one of claims 1 to 12, wherein said cancer is esophageal cancer, preferably Esophageal Squamous Cell Carcinoma (ESCC).

14. The antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer is gastric cancer having a mutation in a gene selected from UGT1A1, UGT1A8 and/or PIK3 CA.

15. The antibody or functional part, derivative and/or analogue thereof for use according to claim 6, wherein the cancer has a mutation in the gene encoding KRAS, preferably wherein the mutation is G12C; the cancer has a mutation in a gene encoding UGT1A1, preferably wherein the mutation is G71R; and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R.

16. The antibody or functional part, derivative and/or analogue thereof for use according to claim 2, wherein the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R and the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R.

17. The antibody or functional part, derivative and/or analogue thereof for use according to claim 10, wherein the cancer further has a mutation in PIK3CA, preferably wherein the mutation is E545K.

18. The antibody or functional part, derivative and/or analogue thereof for use according to any one of claims 1 to 17, wherein the cancer is gastric cancer.

19. An antibody or functional part, derivative and/or analogue thereof for use according to any preceding claim, wherein the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in figure 3; or an amino acid sequence having at most 15, preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and preferably no more than 5, 4, 3, 2 or 1 amino acid modifications, including insertions, deletions, substitutions or combinations thereof, relative to the VH in a VH chain MF3755 as depicted in figure 3; and wherein the VH chain of said variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in figure 3; or an amino acid sequence having up to 15, preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and preferably no more than 5, 4, 3, 2 or 1 amino acid modifications, including insertions, deletions, substitutions or combinations thereof, relative to the VH in a VH chain MF5816 as depicted in figure 3.

20. The antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein said variable domain that binds LGR5 binds an epitope within amino acid residues 21-118 of the human LGR5 sequence depicted in figure 1.

21. The antibody or functional part, derivative and/or analogue thereof for use according to claim 20, wherein amino acid residues at positions 43, 44, 46, 67, 90 and 91 of human LGR5 are involved in the binding of an LGR5 binding variable domain to LGR5.

22. An antibody or a functional part, derivative and/or analogue thereof for use according to claim 20 or 21, wherein the LGR5 binding variable domain binds weakly to an LGR5 protein comprising one or more of the amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A and 91A.

23. The antibody or functional part, derivative and/or analogue thereof for use according to any of the preceding claims, wherein the variable domain that binds EGFR binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence depicted in figure 2.

24. The antibody or functional part, derivative and/or analogue thereof for use according to claim 23, wherein the amino acid residues at positions I462, G465, K489, I491, N493 and C499 of human EGFR are involved in the binding of the EGFR binding variable domain to EGFR.

25. The antibody or functional part, derivative and/or analogue thereof for use according to claim 23 or 24, wherein the EGFR binding variable domain binds weakly to EGFR proteins comprising one or more of the amino acid residue substitutions selected from the group consisting of I462A, G465A, K489A, I491A, N493A and C499A.

26. An antibody or functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein said antibody is enhanced by ADCC.

27. An antibody or a functional part, derivative and/or analogue thereof for use according to any one of the preceding claims, wherein the antibody is non-fucose modified.

28. A method of treating the stomach, esophagus or gastro-esophageal junction comprising administering to a subject in need thereof an antibody or functional part, derivative and/or analogue thereof comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5.

29. The antibody or functional part, derivative and/or analogue thereof or the method for use according to any one of the preceding claims, wherein treatment with said antibody or functional part, derivative and/or analogue thereof is preceded by a step of diagnosing Her2 status of said subject.

30. The antibody or functional part, derivative and/or analogue thereof according to claim 29, wherein diagnosis is testing Her2 status by ISH or IHC.